Modified BDNF

ABSTRACT

Modified BDNF having improved pharmacological activities, pharmacokinetics and physical properties can be obtained by modifying BDNF with a 1-acyl-glycerol derivative. This BDNF being modified with a 1-acyl-glycerol derivative of the present invention has more efficacious and more excellent pharmacokinetic properties with retaining the useful effects being characteristic to BDNF which are useful as remedies for neurodegenerative diseases and diabetes mellitus, and hence, it is particularly useful as a therapeutic agent for treatment of type 2 diabetes mellitus.

This application is the national phase under 35 U.S.C. § 371 of PCTInternational Application No. PCT/JP01/01422 which has an Internationalfiling date of Feb. 26, 2001, which designated the United States ofAmerica.

TECHNICAL FIELD

The present invention relates to a brain-derived neurotrophic factorbeing modified with a 1-acyl-glycerol derivative. In addition, thepresent invention also relates to an agent for treatment of type 2diabetes mellitus, which comprises as the active ingredient abrain-derived neurotrophic factor being modified with a1-acyl-glycerol-derivative.

BACKGROUND ART

One of the neurotrophic factors, a brain-derived neurotrophic factor(hereinafter, occasionally referred to as BDNF), is a protein, which isprovided from target cells or neurons and glial cells and Schwann cellsin the living body, and shows activities to maintain the survival anddifferentiation of neurons. BDNF has been known to act as a specificligand of receptor (trkB), which is a product of p75 and trk genes (cf.,Takeshi NONOMURA, Hiroshi HATANAKA; Jikken Igaku, vol. 13, p. 376(1995)).

BDNF has been known as a therapeutic agent for treatment ofneurodegenerative diseases (e.g., ALS) or diabetic peripheral neuropathy(cf., A. P. Mizisin, et al., Journal of Neuropathology and ExperimentalNeurology, vol.56, p. 1290 (1997)).

In addition, WO 98/32458 discloses that BDNF is useful as a therapeuticagent for treatment of diabetic mellitus.

Since BDNF is a useful protein exhibiting the above mentioned variousfunctions, it has been desired to develop a compound with moreefficacious and more excellent pharmacokinetics with retaining inherentactivities of BDNF.

There has been tried to improve the physical properties and activitiesof proteins with maintaining their functions by modifications. Forexample, JP 2679852 and JP 2718809 disclose common techniques ofmodifying physiologically active proteins with lecithin or lysolecithin,specifically modification of superoxide dismutase (SOD). However, thereis no specific disclosure of modifying BDNF, which is a useful proteinexhibiting various functions.

DISCLOSURE OF INVENTION

An object of the present invention is to provide a compound being moreefficacious with retaining inherent activities of BDNF and furtherhaving an excellent pharmacokinetics.

The present inventors have found that the pharmacological activities,pharmacokinetics and physical properties of BDNF are improved bymodifying BDNF with a 1-acyl-glycerol derivative, and have accomplishedthe present invention.

More particularly, the present invention relates to the following:

(1) A BDNF being modified with a 1-acyl-glycerol derivative:

(2) A modified BDNF according to the above (1), which is the compound ofthe formula (1):

A(X—B)_(m)   (1)

wherein A is a residue of brain-derived neurotrophic factor,

B is a residue of a 1-acyl-glycerol derivative having a hydroxyl groupat the 2-position of the glycerol moiety, which is prepared by removinga hydrogen atom from the hydroxyl group,

X is a chemical cross-linkage, and

m is an average number of the introduction and is not less than about0.5;

(3) A modified BDNF according to the above (2), wherein X is a group ofthe formula (2):

wherein R¹ is an alkylene group,

or a group of the formula (3):

wherein R² and R³ are independently an alkylene group;

(4) A modified BDNF according to the above (2), wherein the1-acyl-glycerol derivative is 1-acyl-glycero-3-phosphoryl choline,1-acyl-glycero-3-phosphoryl serine, or 1-acyl-grycero-3-phosphorylethylamine;

(5) A modified BDNF according to the above (2), wherein B is a1-acyl-glycero-3-phosphoryl choline residue of the formula (4):

wherein R⁴ is an acyl group,

a 1-acyl-glycero-3-phosphoryl serine residue of the formula (5):

wherein R⁴ is an acyl group,

or a 1-acyl-glycero-phosphoryl ethylamine residue of the formula (6):

wherein R⁴ is an acyl group;

(6) A modified BDNF according to the above (2) or (3), wherein B is agroup of the formula (4):

wherein R⁴ is an acyl group;

(7) A modified BDNF according to any one of the above (2), (3), (4), (5)and (6), wherein the acyl group is an alkanoyl group having 8 to 30carbon atoms;

(8) A modified BDNF according to any one of the above (2), (3), (4),(5), (6) and (7), wherein the acyl group is palmitoyl group;

(9) A modified BDNF according to any one of the above (2), (3), (4),(5), (6), (7) and (8), wherein m is in the range of from about 1 toabout 6;

(10) A modified BDNF according to any one of the above (2), (3), (4),(5), (6), (7), (8) and (9), wherein X is a group of the formula (2):

wherein R¹ is an alkylene group;

(11) A modified BDNF according to the above (10), wherein R¹ is astraight chain alkylene group having 2 to 10 carbon atoms;

(12) A modified BDNF according to the above (10), wherein R¹ istrimethylene;

(13) A modified BDNF of the formula (7):

A(X—B)_(n)   (7)

wherein A is a residue of brain-derived neurotrophic factor,

B is a residue of a 1-acyl-glycerol derivative having a hydroxyl groupat the 2-position of the glycerol moiety, which is prepared by removinga hydrogen atom from the hydroxyl group,

X is a chemical cross-linkage, and

n is an integer of 1 or more;

(14) A modified BDNF according to the above (13), wherein X is a groupof the formula (2):

wherein R¹ is an alkylene group, or

or a group of the formula (3):

wherein R² and R³ are independently an alkylene group;

(15) A modified BDNF according to the above (13), wherein B is a1-acyl-glycero-3-phosphoryl choline residue of the formula (4):

wherein R⁴ is an acyl group,

a 1-acyl-glycero-3-phosphoryl serine residue of the formula (5):

wherein R⁴ is an acyl group,

or a 1-acyl-glycero-phosphoryl ethylamine residue of the formula (6):

wherein R⁴ is an acyl group;

(16) A modified BDNF according to the above (13) or (14), wherein B is agroup of the formula (4):

wherein R⁴ is an acyl group;

(15) A therapeutic agent for treatment of type 2 diabetes mellitus,which comprises as the active ingredient a BDNF being modified with a1-acyl-glycerol derivative;

(16) A therapeutic agent for treatment of type 2 diabetes mellituscomprising as the active ingredient a modified BDNF according to theabove (15), which is a compound of the formula (1):

A (X—B)_(m)   (1)

wherein A is a residue of brain-derived neurotrophic factor,

B is a residue of a 1-acyl-glycerol derivative having a hydroxyl groupat the 2-position of the glycerol moiety, which is prepared by removinga hydrogen atom from the hydroxyl group,

X is a chemical cross-linkage, and

m is an average number of the introduction and is not less than about0.5;

(17) A therapeutic agent for treatment of type 2 diabetes mellituscomprising as the active ingredient a modified BDNF according to theabove (16), wherein X is a group of the formula (2):

wherein R¹ is an alkylene group,

or a group of the formula (3):

wherein R² and R³ are independently an alkylene group;

(18) A therapeutic agent for treatment of type 2 diabetes mellituscomprising as the active ingredient a modified BDNF according to theabove (16), wherein the 1-acyl-glycerol derivative is1-acyl-glycero-3-phosphoryl choline, 1-acyl-glycero-3-phosphoryl serine,or 1-acyl-grycero-3-phosphoryl ethylamine;

(19) A therapeutic agent for treatment of type 2 diabetes mellituscomprising as the active ingredient a modified BDNF according to theabove (16), wherein B is a 1-acyl-glycero-3-phosphoryl choline residue

wherein R⁴ is an acyl group,

a 1-acyl-glycero-3-phosphoryl serine residue of the formula (5):

wherein R⁴ is an acyl group,

or a 1-acyl-glycero-phosphoryl ethylamine residue of the formula (6):

wherein R⁴ is an acyl group;

(20) A therapeutic agent for treatment of type 2 diabetes mellituscomprising as the active ingredient a modified BDNF according to theabove (16) or (17), wherein B is a group of the formula (4):

wherein R⁴ is an acyl group;

(21) A therapeutic agent for treatment of type 2 diabetes mellituscomprising as the active ingredient a modified BDNF according to any oneof the above (16), (17), (18), (19) and (20), wherein the acyl group isan alkanoyl group having 8 to 30 carbon atoms;

(22) A therapeutic agent for treatment of type 2 diabetes mellituscomprising as the active ingredient a modified BDNF according to any oneof the above (16), (17), (18), (19), (20) and (21), wherein the acylgroup is palmitoyl group;

(23) A therapeutic agent for treatment of type 2 diabetes mellituscomprising as the active ingredient a modified BDNF according to any oneof the above (16), (17), (18), (19), (20), (21) and (22), wherein m isin the range of from about 1 to about 6;

(24) A therapeutic agent for treatment of type 2 diabetes mellituscomprising as the active ingredient a modified BDNF according to any oneof the above (16), (17), (18), (19), (20), (21), (22) and (23), whereinX is a group of the formula (2):

wherein R¹ is an alkylene group;

(25) A therapeutic agent for treatment of type 2 diabetes mellituscomprising as the active ingredient a modified BDNF according to theabove (24), wherein R¹ is a straight chain alkylene group having 2 to 10carbon atoms;

(26) A therapeutic agent for treatment of type 2 diabetes mellituscomprising as the active ingredient a modified BDNF according to theabove (25), wherein R¹ is trimethylene;

(27) A therapeutic agent for treatment of type 2 diabetes mellituscomprising as the active ingredient a modified BDNF, which is thecompound of the formula (7):

A(X—B)_(n)   (7)

wherein A is a residue of a brain-derived neurotrophic factor,

B is a residue of a 1-acyl-glycerol derivative having a hydroxyl groupat the 2-position of the glycerol moiety, which is prepared by removinga hydrogen atom from the hydroxyl group,

X is a chemical cross-linkage, and

n is an integer of 1 or more;

(28) A therapeutic agent for treatment of type 2 diabetes mellituscomprising as the active ingredient a modified BDNF according to theabove (27), wherein X is a group of the formula (2):

wherein R¹ is an alkylene group,

or a group of the formula (3):

wherein R² and R³ are independently an alkylene group.

(29) A therapeutic agent for treatment of type 2 diabetes mellituscomprising as the active ingredient a modified BDNF according to theabove (27), wherein B is a 1-acyl-glycero-3-phosphoryl choline residueof the formula (4):

wherein R⁴ is an acyl group,

a 1-acyl-glycero-3-phosphoryl serine residue of the formula (5):

wherein R⁴ is an acyl group,

or a 1-acyl-glycero-phosphoryl ethylamine residue of the formula (6):

wherein R⁴ is an acyl group;

(30) A therapeutic agent for treatment of type 2 diabetes mellituscomprising as the active ingredient a modified BDNF according to theabove (27) or (28), wherein B is a group of the formula (4):

wherein R⁴ is an acyl group.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 indicates a spectrum pattern of time-of-flight mass spectrometry(TOF-MALDI MS) of the lecithinized brain-derived neurotrophic factorobtained in Example 1 and a brain-derived neurotrophic factor.

FIG. 2 is a graph showing the effects of the lecithinized BDNF and aBDNF on the blood glucose level of db/db mice.

FIG. 3 is a graph showing the effects of the lecithinized BDNF and aBDNF on the amount of food consumption of db/db mice.

FIG. 4 is a graph showing the effects of the lecithinized brain-derivedneurotrophic factor and a brain-derived neurotrophic factor on the bodyweights of db/db mice.

BEST MODE FOR CARRYING OUT THE INVENTION

A brain-derived neurotrophic factor being modified with a1-acyl-glycerol derivative of the present invention (hereinafter,occasionally abbreviated as a modified BDNF) can be used as atherapeutic agent for treatment of nervous system disorders anddiseases, or treatment of diabetes mellitus. More particularly, thebrain-derived neurotrophic factor being modified with a 1-acyl-glycerolderivative of the present invention can be administered to patientshaving nervous system injured by wound, surgery, ischemia, infection,metabolic diseases, malnutrition, malignant tumor, or toxic drug, etc.Especially, it can be used in the treatment of conditions whereinsensory neurons or retinal ganglion cells are injured. More especially,the modified BDNF of the present invention can be used in the treatmentof congenital conditions or neurodegenerative diseases, for example,Alzheimer's disease, Parkinson's disease (the symptoms of Parkinson'sdisease may be caused by the degeneration of dopaminergic neuron),Parkinson-Plus syndromes (e.g., progressive spranuclear palsy(Steele-Richardson-Olszewski syndromes), olivopontocerebellar atrophy(OPCA), Shy-Drager syndromes (Multiple Systems Atrophy), and Parkinsondementia complex of Guam), and Huntington's chorea, but are not limitedthereto. Further, the present modified BDNF can be used in the treatmentof sensory nerve dysfunction and congenital diseases orneurodegenerative diseases being associated with degenerative of retina.In addition, the present modified BDNF can be used in the treatment ofinherited convulsive paraplegia associated with retina degeneration(Kjellin and Barnard-Scholz syndromes), retinitis pigmentosa, Stargardtdisease, Usher syndromes (retinitis pigmentosa accompanied by congenitalhearing loss) and Refsum syndrome (retinitis pigmentosa, congenitalhearing loss, and polyneuropathy).

The present modified BDNF can also be used in order to accelerate therecovery of a patient of diabetic neuropathy or multiple mononeuropathy.Further, the present modified BDNF can also be used as a therapeuticagent for treatment of diabetes mellitus, especially type 2 diabetesmellitus having a hyperglycemia due to insulin dysfunction or insulinresistance, etc.

In addition, the present modified BDNF can be used as an agent forameliorating insulin dysfunction or insulin resistance, or an agent fortreating or ameliorating hyperglycemia accompanied by reduction orfailure of insulin secretion, or an agent for treating or amelioratinghyperlipemia, or an agent for treating or ameliorating hyperinsulinemia.

The groups of the formula (1), the formula (2), the formula (3), theformula (4), the formula (5) and the formula (6) are explained in moredetail.

The residue of a brain-derived neurotrophic factor as represented by Ameans a residue being prepared by removing m or n numbers of hydroxylgroups or hydrogen atoms to which a group of (X—B)_(m) or (X—B)_(n)bound, from the functional groups such as carboxyl groups and aminogroups of an amino acid (e.g., lysine residue) of BDNF.

The average number of the introduction of modifying groups means theaverage number of modifying groups being introduced into one molecule ofBDNF, and is not less than 0.5, preferably about 1 or more, morepreferably in the range of about 1 to about 10, especially in the rangeof about 1 to about 6. Most preferably, it is in the range of about 2 toabout 4.

The integer of 1 or more for n is, for example, an integer of 1 to 10,preferably an integer of 1 to 6, more preferably an integer of 2 to 4.

The alkylene group includes a straight chain or branched chain alkylenegroup having 1 to 24 carbon atoms, preferably a straight chain alkylenegroup having 2 to 10 carbon atoms, for example, ethylene, trimethylene,tetramethylene, pentamethylene, hexamethylene, heptamethylene,octamethylene, nonamethylene, decamethylene, etc.

The acyl group includes, for example, an alkanoyl group, an alkenoylgroup, an aroyl group, a substituted aroyl group, etc.

The alkanoyl group includes an alkanoyl group having 8 to 30 carbonatoms, for example, octanoyl, nonanoyl, decanoyl, undecanoyl,dodecanoyl, tridecanoyl, tetradecanoyl, pentadecanoyl, hexadecanoyl,heptadecanoyl, octadecanoyl, nonadecanoyl, eicosanoyl, heneicosanoyl,tricosanoyl, tetracosanoyl, pentacosanoyl, hexacosanoyl, heptacosanoyl,octacosanoyl, nonacosanoyl, triacontanoyl, etc., and preferably analkanoyl group having 14 to 22 carbon atoms such as tetradecanoyl,pentadecanoyl, hexadecanoyl, heptadecanoyl, octadecanoyl, nonadecanoyl,eicosanoyl, heneicosanoyl, etc.

The aroyl group includes an aroyl group having 7 to 11 carbon atoms, forexample, benzoyl group, naphthoyl group, etc.

The alkenoyl group includes an alkenoyl group having 8 to 30 carbonatoms, for example, oleoyl, linoleoyl, arachidonoyl, etc.

The substituted aroyl group includes an aroyl group being substituted byan alkyl group, a halogen atom, an alkoxy group, a hydroxyl group, anitro group, etc., for example, 4-methylbenzoyl, 2-methylbenzoyl,4-fluorobenzoyl, 2-fluorobenzoyl, 4-chlorobenzoyl, 3-chlorobenzoyl,2-chlorobenzoyl, 4-methoxybenzoyl, 3-methoxybenzoyl,2-methoxybenzoyl,4-hydroxybenzoyl, 3-nitrobenzoyl, etc.

The alkyl group includes a lower alkyl group having 1 to 6 carbon atoms,etc., for example, methyl, ethyl, propyl, butyl, pentyl, hexyl,2-methylpropyl, etc.

The halogen atom is fluorine atom, chlorine atom, bromine atom, andiodine atom.

The alkoxy group includes a lower alkoxy group having 1 to 6 carbonatoms, etc., for example, methoxy, ethoxy, propyloxy, butyloxy,pentyloxy, hexyloxy, 2-methylpropyloxy, etc.

The chemical cross-linkage for X is a chemical cross-linkage consistingof an organic group combining A and B after the chemical cross-linkingreaction. One end of the chemical cross-linkage is preferably bound tothe 1-acyl-glycerol derivative via an ester bond. The other end of thechemical cross-linkage is directly bound to a functional group such asan amino group or a carboxyl group of the brain-derived neurotrophicfactor. The chemical cross-linkage preferably has a carboxyl group, anamino group, a hydroxyl group, an imino group, etc. as a functionalgroup. In addition, the chemical cross-linkage may optionally has two ormore functional groups, and one of the functional groups is bound to the1-acyl-glycerol derivative and other functional group is bound to afunctional group of the brain-derived neurotrophic factor. The chemicalcross-linkage may further intervene between other functional groups.Preferable chemical cross-linkage is, for example, a group of theformula (2):

wherein R¹ is an alkylene group,

a group of the formula (3):

wherein R² and R³ are independently an alkylene group, and the like.More preferable chemical cross-linkage is a group of the formula (2′:

wherein R^(1 ′) is a C₂-C₁₀ alkylene group.

In the integer of 1 or more for n, preferable integer is in the range of1 to 10, and more preferable integer is in the range of 1 to 5.

The brain-derived neurotrophic factor (BDNF) of the present inventionincludes modified ones prepared by deletion of a part of amino acidsequence, or a substitution by other amino acid(s), or an addition of apart of other amino acid sequence, or ones having one or more aminoacids bound at the N-terminus and/or the C-terminus, or ones wherein thesugar chain is deleted or substituted, as far as they exhibitsubstantially the same activity as naturally occurred brain-derivedneurotrophic factor.

The BDNF used in the present invention can be prepared by variousmethods, for example, by cultivating a primary culture cell or anestablished cell line that can produce the neurotrophic factor, andisolating and purifying it from the culture medium thereof (e.g.,culture supernatant, cultured cells). Moreover, a recombinantneurotrophic factor can be obtained by a conventional gene engineeringtechnique, e.g., by inserting a gene encoding a BDNF into a suitablevector, transforming a suitable host with the recombinant vector, andisolating from a culture supernatant of the resulting transformant. Thehost cells to be used in the above process are not limited, and may beany conventional host cells which have been used in a gene engineeringtechnique, for example, Escherichia coli, Bacillus subtilis, yeasts,mold fungi, plant cells or animal cells. For example, when aconventional gene engineering technique is employed, BDNF is prepared byinserting a gene encoding BDNF into a suitable vector, transforming asuitable host with the recombinant vector, and isolating it from aculture supernatant of the resulting transformant (cf., Prof. Natl.Acad. Sci. USA, vol. 88, p. 961 (1991); Biochem. Biophys. Res. Commun.,vol. 186, p. 1533 (1992)). The gene engineering technique is suitablefor production of BDNF of same quality in a large scale.

Method for Production

wherein A is a residue of a brain-derived neurotrophic factor, B is aresidue of 1-acyl-glycerol derivative having a hydroxy group at the2-position of the glycerol moiety, which is prepared by removing ahydrogen atom from the hydroxyl group, X is a chemical cross-linkage, mis an average number of the introduction, and is not less than about0.5, and X ¹ is a chemical cross-linkage having a reactive functionalgroup.

The reactive functional group for X¹ means a functional group beingactivated so as to proceed the chemical cross-linking reaction with afunctional group such as an amino group, a carboxyl group of BDNF. Moreparticularly, the reactive functional group is, for example, a carboxylgroup, an amino group, or an activated carboxyl group.

For compound (11) having a carboxyl group as a reactive functionalgroup, the reactive functional group may be introduced by reacting adicarboxylic acid anhydride having 2 carboxyl groups with a1-acyl-glycerol derivative in an inert solvent in the presence of acatalyst, if necessary, in the presence of a base. The inert solventincludes, for example, aprotic solvents (e.g., dimethylformamide,acetonitrile, etc.), ethers (e.g., tetrahydrofuran, etc.), aromaticsolvents (e.g., toluene, etc.), halogenated hydrocarbons (e.g.,dichloromethane, dichloroethane, etc.), etc. The catalyst includes, forexample, acids (e.g., p-toluenesulfonic acid, etc.), pyridines (e.g.,4-dimethylaminopyridine, 4-(N-pyrrolidyl)-pyridine, etc.). The baseincludes, for example, organic bases (e.g., triethylamine, pyridine,etc.). The reaction temperature is in the range of about 0° C. to about100° C.

For compound (11) having an activated carboxyl group as a reactivefunctional group, the method for activation of a carboxyl group is, forexample, mixed acid anhydride method, active ester method, etc. Themixed acid anhydride method is carried out by reacting a 1-acyl-glycerolderivative having a carboxyl group being introduced thereto obtained inthe above method with an alkyloxycarbonyl halide in an inert solvent inthe presence of a base under non-aqueous conditions to give the compound(11). The alkyloxycarbonyl halide includes, for example,isobutyloxycarbonyl chloride, sec-butyloxycarbonyl chloride,isopropyloxycarbonyl chloride, ethyloxycarbonyl chloride,methyloxycarbonyl chloride, etc. The inert solvent may be aproticsolvents (e.g., dimethylformamide, acetonitrile, etc.), ethers (e.g.,tetrahydrofuran, etc.), aromatic solvents (e.g., toluene, etc.), etc.The base may be alkylamines (e.g., triethylamine, N-methylmorpholine,etc.), pyridines (e.g., pyridine, 4-dimethylaminopyridine,4-(N-pyrrolidinyl)pyridine, etc.), and a combination of these solvents.The reaction temperature is in the range of about −20° C. to about roomtemperature.

The active ester method is carried out by reacting a 1-acyl-glycerolderivative having a carboxyl group introduced thereto obtained in theabove method and an N-hydroxyimide or a phenol in the presence of acondensing agent in an inert solvent in the presence of a base to givethe compound (11). The N-hydroxyimide includes, for example,N-hydroxysunccinimide, etc. The phenol includes, for example,4-nitro-phenol, pentachlorophenol, pentafluorophenol, 4-nitorthiophenol,etc. The condensing agent includes carbodiimides such asdicyclohexylcarbodiimide,1-(3-dimethylamino)propyl)-3-ethyl-carbodiimide hydrochloride, etc. Theinert solvent may be aprotic solvents (e.g., dimethyl-formamide,acetonitrile, etc.), ethers (e.g., tetrahydrofuran, etc.), aromaticsolvents (e.g., toluene, etc.), halogenated hydrocarbons (e.g.,dichloromethane, dichloroethane, etc.). The base may be, for example,alkylamines (e.g., triethylamine, etc.), pyridines (e.g., pyridine,4-dimethylaminopyridine, 4-(N-pyrrolidyl)pyridine, etc.), or acombination of these solvents. The reation temperature is in the rangeof about 0° C. to about 100° C.

In case of using compound (11) having a carboxyl group as a reactivefunctional group, a BDNF (1) being modified with a 1-acyl-glycerolderivative can be prepared by reacting the compound (11) with BDNF usinga condensing agent in a solvent in the presence of a base. Thecondensing agent includes carbodiimides such asdicyclohexyl-carbodiimide,1-(3-dimethylamino)propyl)-3-ethyl-carbodiimide hydrochloride, etc., ora combination of these carbodiimides and an N-hydroxyimide such asN-hydroxysuccinimide, etc. The solvent is, for example, aprotic solvents(e.g., dimethylformamide, acetonitrile, etc.), ethers (e.g.,tetrahydrofuran, etc.), or a mixed solvent of these solvents and water,and water, etc. The base may be, for example, alkylamines (e.g.,triethylamine, etc.), pyridines (e.g., pyridine,4-dimethylaminopyridine, 4-(N-pyrrolidyl)pyridine, etc.) and acombination thereof. The reaction temperature is in the range of about0° C. to about 100° C.

In case of using compound (11) having an activated carboxyl group as areactive functional group, a BDNF (1) being modified with a1-acyl-glycerol derivative can be prepared by reacting the compound (11)with BDNF in an inert solvent in the presence of a base. The inertsolvent is, for example, aprotic solvents (e.g., dimethylformamide,acetonitrile, etc.), ethers (e.g., tetrahydrofuran, etc.), or a mixedsolvent of these solvents and water, and water, etc. The base may be,for example, alkylamines (e.g., triethylamine, etc.), pyridines (e.g.,pyridine, 4-dimethylaminopyridine, 4-(N-pyrrolidyl)pyridine, etc.) and acombination thereof. The reaction temperature is in the range of about0° C. to about 100° C.

In case of using compound (11) having an activated imide group as areactive functional group, a BDNF (1) being modified with a1-acyl-glycerol derivative can be prepared by reacting the compound (11)with BDNF in an inert solvent in the presence of a base. The inertsolvent may be, for example, aprotic solvents (e.g., dimethylformamide,acetonitrile, etc.), ethers (e.g., tetrahydrofuran, etc.), a combinationof water and these solvent, or water. The base includes, for example,alkylamines (e.g., triethylamine, etc.), pyridines (e.g., pyridine,4-dimethylaminopyridine, 4-(N-pyrrolidyl)pyridine, etc.), or acombination of these solvents. The reaction temperature is in the rangeof about 0° C. to about 100° C.

The BDNF being modified with a 1-alkanoyl-glycerol derivative of thepresent invention which is obtained by the above method can be purifiedby one of the following methods or a combination thereof. (1)Polyethylenimine fractionation, (2) Gel filtration chromatography withSephacryl S-200; (3) Ion exchange chromatography with Biorex-70 resin orCM Sephadex; (4) Ammonium sulfate fractionation and/or pH fractionation,and (5) Affinity chromatography using an antibody resin which isprepared from an antibody isolated from hybridomas or sensitizedanimals. The modified BDNF can be desorbed under acidic conditions orslightly denaturation conditions.

The exact dosage and the administration schedule of the brain-derivedneurotrophic factor being modified with a 1-alkanoyl-glycerol derivativeof the present invention should vary according to the dosage to berequired for each patient, the method for treatment, kinds of diseasesto be treated, or the degree of necessity, and further according to thediagnosis by a physician. When administered parenterally, the dosage andthe frequency of the administration may vary according to theconditions, ages, body weights of patients, and administration routes,but when it is administered subcutaneously or intravenously in the formof an injection, then the daily dosage thereof is in the range of about1 to about 2500 μg, preferably in the range of about 10 to about 500 μg,per 1 kg of the body weight in an adult. When it is administered to theair tract in the form of an aerosol spray, the daily dosage thereof isin the range of about 1 μg to about 2500 μg, preferably in the range ofabout 10 to about 500 μg, per 1 kg of the body weight in an adult. Theadministration schedule is either continuous daily administration,intermittent administration, or a schedule of combining these methods.

When administered orally, the dosage and the frequency of administrationmay vary according to the conditions, ages, body weights of patients,and administration routes, and the daily dosage thereof is in the rangeof about 5 to about 2500 μg, preferably in the range of about 10 toabout 1000 μg per 1 kg of the body weight in an adult.

The present modified BDNF is locally administered to severed sensoryneurons at one of the various tissues such as geniculate, pyramidal andnodule nerve ganglion; vestibular auditory nerve complex of the 8thcranial nerve; ventral posterolateral nucleus of maxillomandibular oftrigeminal ganglion; and mesencephalic nucleus of trigeminal nerve. Inthis case, it is preferably administered by absorbing to a membranebeing capable of transplating at neighborhood of said severed neurons,such as Silastic membrane.

The exact dosage and the administration schedule of the brain-derivedneurotrophic factor being modified with a 1-alkanoyl-glycerol derivativeof the present invention for a patient suffering from type 2 diabetesmellitus should vary according to the dosage to be required for eachpatient, the method for treatment, kinds of diseases to be treated, orthe degree of necessity, and further according to the diagnosis by aphysician. When administered parenterally, the dosage and the frequencyof the administration may vary according to the conditions, ages, bodyweights of patients, and administration routes, but when it isadministered subcutaneously or intravenously in the form of aninjection, then the daily dosage thereof is in the range of about 1 toabout 500 μg, preferably in the range of about 10 to about 250 μg, per 1kg of the body weight in an adult. When it is administered to the airtract in the form of an aerosol spray, the daily dosage thereof is inthe range of about 1 μg to about 500 μg, preferably in the range ofabout 10 to about 250 μg, per 1 kg of the body weight in an adult. Theadministration schedule is either continuous daily administration,intermittent administration, or a schedule of combining these methods.

A pharmaceutical composition can be prepared by mixing the brain-derivedneurotrophic factor being modified with a 1-acyl-glycerol derivative ofthe present invention with a pharmaceutically acceptable non-toxiccarrier. When a pharmaceutical composition for parenteral administration(subcutaneous injection, intramuscular injection, or intravenousinjection) is prepared, it is preferably in the form of a solutionpreparation or a suspension preparation. When a pharmaceuticalcomposition for intravaginal administration or rectal administration isprepared, it is preferably in the form of a semi-solid preparation suchas cream or suppository. When a pharmaceutical composition forintranasal administration is prepared, it is preferably in the form of apowder, a nasal drop, or an aerosol.

The pharmaceutical composition is administered in the form of a singledosage unit, and can be prepared by any conventional method that isknown in the pharmaceutical field such as methods disclosed inRemington's Pharmaceutical Science (published by Mack PublishingCompany, Easton, Pa., 1970). An injection preparation may optionallycontain as a pharmaceutical carrier a protein derived from plasma suchas albumin, an amino acid such as glycine, or a carbohydrate such asmannitol, and additionally a buffering agent, a solubilizer, or anisotonic agent, etc. can be contained. When the present pharmaceuticalcomposition is in the form of an aqueous solution preparation or alyophilized preparation, it may preferably contain a surfactant such asTween 80 (registered trade mark), Tween 20 (registered trade mark), etc.in order to avoid aggregation. When the present pharmaceuticalcomposition is a composition for parenteral administration other than aninjection preparation, then it may contain distilled water orphysiological saline solution, polyalkylene glycol such as polyethyleneglycol, an oil derived from plant, hydrogenated naphthalene, etc. Forexample, a pharmaceutical composition such as a suppository forintravaginal administration or rectal administration may contain as aconventional excipient polyalkylene glycol, vaseline, cacao butter, etc.A pharmaceutical composition for intravaginal administration may containan absorbefacient such as a bile salt, an ethylenediamine salt, acitrate, etc. A pharmaceutical composition for inhalation may be in theform of a solid preparation, and may contain as an excipient lactose,etc., and a pharmaceutical composition for intranasal drop may be in theform of an aqueous solution or an oily solution.

The present pharmaceutical composition is especially preferable in theform of a formulation by which the present compound can persistently begiven to a subject by a single administration for a long term, e.g., forone week to one year, and various sustained release preparations, depotpreparations, or implant preparations can be employed.

The present BDNF being modified with a 1-acyl-glycerol derivative may bein the form of a pharmaceutically acceptable salt thereof having a lowsolubility in a living fluid. Such pharmaceutically acceptable saltsare, for example, (1): an acid addition salt such as phosphate, sulfate,citrate, tartrate, tannate, pamoate, alginate, polyglutamate,naphthalenemono- or di-sulfonate, polygalacturonate, etc., (2): a saltor complex with a polyvalent metal cation such as zinc, calcium,bismuth, barium, nickel, etc, or a combination of (1) and (2), forexample, a tannic acid-zinc salt, etc. The present BDNF being modifiedwith a 1-acyl-glycerol derivative is preferably converted into aslightly-water-soluble salt thereof, which is mixed with a gel, forexample, aluminum monostearate gel and sesame oil, etc. to give asuitable injection preparation. In this case, especially preferable saltis a zinc salt, a tannic acid-zinc salt, a pamoate, etc. Another type ofa sustained release injection preparation is ones wherein the presentBDNF being modified with a 1-acyl-glycerol derivative is preferablyconverted into a slightly-water-soluble salt thereof, which is furtherenclosed in a slow-disintegrative non-toxic and non-antigenic polymersuch as a polymer or a copolymer of polylactic acid/polyglycolic acid.In this case, especially preferable salt is zinc salt, tannic acid-zincsalt, pamoate, etc. In addition, a neurotrophic factor or aslightly-water-soluble salt thereof can be enclosed into a cholesterolmatrix or a collagen matrix to give a sustained release preparation.

The pharmaceutical preparation for oral administration may be ones whichare prepared by microencapsulating the present BDNF being modified witha 1-acyl-glycerol derivative or a salt thereof with lecithin,cholesterol, a free fatty acid, or ones which are prepared by enclosingsaid microcapsules into gelatin capsules, or ones which are prepared byenclosing the BDNF being modified with a 1-acyl-glycerol derivative or asalt thereof in enteric capsules, etc. These preparations mayadditionally contain, for example, an absorbefacient, a stabilizer, asurfactant, etc.

Since lecithin, which is one of the 1-acyl-glycerol derivatives used formodifying BDNF of the present invention, is a non-toxic substance beingwidely and naturally existing, lecithin-modified BDNF is also safe fromthe viewpoint of toxicity.

EXAMPLES Reference Example 1

Synthesis of 2-(4-hydroxycarbonylbutyroyl)lysolecithin:

To a suspension of lysolecithin, which has a hydroxy group at the2-position of the glycerol moiety (the acyl group R⁴ of the formula (4)is palmitoyl group, hereinafter the same) (204 mg, 0.4 mmol) inchloroform-pyridine (8 ml/2 ml) were added DMAP(N,N-dimethylaminopyridine) (98 mg, 0.8 mmol) and glutaric anhydride (91mg, 0.8 ml), and the mixture was stirred at 60° C. for 15 hours. Thereaction solution was cooled and concentrated under reduced pressure.The concentrated residue was dissolved inchloroform/methanol/water=4:5:1 (2 ml), and the reaction solution waspassed through the ion exchange column (Dowex 50W—X8), which had beenequilibrated with said solution, concentrated under reduced pressure,and the residue was purified by silica gel column chromatography. Yield:225 mg (0.36 mmol, 90%) ¹H-HMR (CDCL₃): 0.84 (t, 3H), 1.20 (brs),1.52-1.60 (brs, 2H), 1.80-1.95 (m, 2H), 2.20-2.42 (m, 6H), 3.35 (s, 9H),3.78 (m, 4H), 3.90-4.35 (m, 4H), 5.20 (s, 1H)

Reference Example 2

Synthesis of an Active Ester of2-(4-hydroxycarbonylbutyroyl)-lysolecithin:

The carboxylic acid (225 mg, 0.36 mmol) obtained in Reference Example 1was dissolved in dichloromethane (5 ml), and the mixture was cooled to0° C., and thereto were added N-hydroxysuccinimide (41 mg, 0.36 mmol)and DCC (74 mg, 0.36 mmol). The mixture was stirred at room temperaturefor 15 hours. The insoluble materials are filtered through Celite, andthe solvent was evaporated under reduced pressure to give an activeester.

Example 1

Water (5.4 ml) was added to a 30 mg/ml solution of r-h BDNF (1.7 ml),and the pH value of the solution was adjusted to pH 11 underwater-cooling, and stirred. The aqueous solution (2.4 ml) of 0.6equivalent of 2-O-(4-hydroxycarbonylbutyryl)lysophosphoryl cholinehydroxysuccinimide ester to the whole amino groups of the r-h BDNF(recombinant human brain-derived neurotrophic factor) was added dropwiseto the reaction mixture, and the mixture was stirred for 12 hours underwater-cooling. The reaction solution was filtered through an ultrafiltration membrane (cut off: molecular weight 10,000) to remove theunreacted agents and lower molecular weight substances. The solvent wasdisplaced with a 10 mM phosphoric acid/potassium phosphate mixedsolution to give a title compound. The average number of theintroduction of the lecithin derivative into the r-h BDNF was measuredby time-of-flight mass spectrometry (TOF-MALDI MS). The spectralpatterns of r-h BDNF and the above compound are shown in FIG. 1.

Example 2

Water (0.7 ml) was added to a 30 mg/ml solution of r-h BDNF (0.25 ml),and the pH value of the solution was adjusted under water-cooling, andthe whole volume was adjusted to 1.2 ml and then stirred. The aqueoussolution (0.23 ml) of 2-0-(4-hydroxycarbonylbutyryl)-lysophosphorylcholine hydroxysuccinimide ester of an equivalent to the whole aminogroups of the r-h BDNF (as shown in the following Table) was addeddropwise to the reaction mixture, and the mixture was allowed to standfor 12 hours under water-cooling. The reaction solution was filteredthrough an ultra filtration membrane (cut off: molecular weight 10,000)to remove the unreacted agents and lower molecular weight substances.The solvent was displaced with a 10 mM phosphoric acid/potassiumphosphate mixed solution to give a title compound. The average number ofthe introduction of the lecithin derivative into the r-h BDNF wasmeasured by time-of-flight mass spectrometry (TOF-MALDI MS).

TABLE 1 pH value of the Equivalents to the reaction solution aminogroups Compound 2 11 0.6 Compound 3 11 0.6 Compound 4 11 0.6 Compound 511 0.6 Compound 6 11 0.6 Compound 7 11 1.5 Compound 8 7 1.5

Example 3

Evaluation of Efficacy of BDNF and Lecithinized BDNF (Example 1) onDiabetes Mellitus, and Study of the Method for Administration and DosageThereof:

Materials and Methods for Experiments

(1) Test animals:

Male C57BL/Ks j-db/db Jcl mice (7 weeks old) were purchased from CleaJapan, Inc. After pre-feeding for 5 weeks, the animals were used in theexperiment at 12 weeks old.

(2) Breeding conditions:

The mice were kept in a room being controlled at a temperature of 23±2°C. under a relative humidity of 55±10% with an illumination cycle oflight on (8:00 to 20:00) and light off (20:00 to 8:00). During thepre-feeding and the experiment, the animals were given food (CE-2, CleaJapan, Inc.) and sterilized tap water ad libitum.

(3) Identification of individuals and cages

Each mouse was identified by writing the number thereof on the tail withoil-based ink. Cages were marked with a label wherein a name in chargeof the experiment, the date of arrival, strain of mice, sex, and sourcewere indicated. During the pre-bleeding, the mice were kept in 10animals/cage. After the experiment started, each animal was keptseparately in 1 animal/cage.

(4) Determination of dosage and grouping

The following groups were set with respect to the dosage of BDNF, andlecithinized BDNF.

1: BDNF 10 mg/kg/day (n=5)

2: BDNF 20 mg/kg/day (n=5)

3: Lecithinized BDNF 1 mg/kg/day (n=5)

4: Lecithinized BDNF 3 mg/kg/day (n=5)

5: Lecithinized BDNF 10 mg/kg/day (n=5)

6: Vehicle-treated group (n=5)

(5) Selection of hyperglycemic animals and grouping thereof

Five days prior to the administration, the mice were kept separately,and the amount of food consumption, body weight, blood glucose levelwere monitored until the administration day. The animal were groupedinto 6 groups as indicated in the above (4) with respect to body weightand blood glucose level on the administration day, and the amount offood consumption (g/head/day) in a period from 5 days before theadministration to the administration day. When grouping, mice having nohyperglycemia (300 mg/kg or less) and mice having apparently in badconditions were excluded in the experiment.

(6) Preparation of dosing solution

The method for preparation is indicated below. The vehicle was 10 mMKH₂PO₄/H₃PO₄ (pH 3.0) containing 0.1% BSA, and the test 5 compound wasdiluted.

TABLE 2 Groups Concentration of dosing solution BDNF 10 mg/kg/Day BDNF1.0 mg/ml BDNF 20 mg/kg/Day BDNF 2.0 mg/ml Lecithinized BDNFLecithinized BDNF 0.1 mg/ml 1 mg/kg/Day Lecithinized BDNF LecithinizedBDNF 0.3 mg/ml 3 mg/kg/Day Lecithinized BDNF Lecithinized BDNF 1.0 mg/ml10 mg/kg/Day

(7) Administration method

The administration was carried out by subcutaneous administration on theback at a dose of 10 ml/kg for 5 days.

(8) Evaluation items

Every day during the administration period, and occasionally during theperiod after and before the administration, the body weight, the amountof food consumption and the blood glucose level were measured. The bodyweight and the amount of food consumption were measured with anautomatic balance, and the blood glucose level was measured by AntosenseII (Sankyo-Bayer) on the blood taken from the tail vein.

(9) Statistics Analysis

Dunnet's test was carried out on the body weight, the amount of foodconsumption, the blood glucose level, comparing with the data of thevehicle-treated group as control at each point by using statisticalprogram SAS (registered trade name). The significant level was set at5%.

(10) Results

The lecithinized BDNF or BDNF was subcutaneously administered to db/dbmice during Day 0 to Day 4 (i.e., from the day of the beginning of theexperiment to the 4th day). The blood glucose level during theadministraion period and after the administration were measured withtime-lapse, and the change of the blood glucose level is indicated inFIG. 2. In FIG. 2, ▪ shows the data of the vehicle-treated group; □shows the data of the BDNF 10 mg/kg treated group; Δ shows the data ofthe BDNF 20 mg/kg treated group; ♦ shows the data of the lecithinizedBDNF 1 mg/kg treated group; ◯ shows the data of the lecithinized BDNF 3mg/kg treated group;  shows the data of the lecithinized BDNF 10 mg/kgtreated group; each data is expressed in the average ±S.D. (n=5);*:P<0.05,**:P<0.01 vs. the vehicle-treated group (Dunnett's test).

As is shown in said FIG. 2, the lecithinized BDNF showed remarkabledose-dependent hypoglycemic activity. Although BDNF showeddose-dependent hypoglycemic activity as well, there was no significantdifference at the dose of 10 mg/kg. In addition, when comparing the dataof the lecithinized BDNF 10 mg/kg treated group and the BDNF 10 mg/kgtreated group, the lecithinized BDNF showed more potent effects.

In addition, after subcutaneously administering a lecithinized BDNF andBDNF to db/db mice during the period of from Day 0 to Day 4, the amountof food consumption was measured during the administration period andafter the administration with time-lapse, and the change of amount offood consumption is indicated in FIG. 3. In FIG. 3, ▪ shows the data ofthe vehicle-treated group; □ shows the data of BDNF 10 mg/kg treatedgroup; Δ shows the data of the BDNF 20 mg/kg treated group; ♦ shows thedata of the lecithinized BDNF 1 mg/kg treated group; ◯ shows the data ofthe lecithinized BDNF 3 mg/kg treated group;  shows the data of thelecithinized BDNF 10 mg/kg treated group; each data is expressed in theaverage ±S.D. (n=5); *:P<0.05,**:P<0.01 vs. the vehicle-treated group(Dunnett's test).

Further, after subcutaneously administering a lecithinized BDNF and BDNFto db/db mice during the period of from Day 0 to Day 4, the body weightwas measured during the administration period and after theadministration with time-lapse, and the change of the body weight isindicated in FIG. 4. In FIG. 4, ▪ shows the data of the vehicle-treatedgroup; □ shows the data of the BDNF 10 mg/kg treated group; Δ shows thedata of the BDNF 20 mg/kg treated group; ♦ shows the data of thelecithinized BDNF 1 mg/kg treated group; ◯ shows the data of thelecithinized BDNF 3 mg/kg treated group;  shows the data of thelecithinized BDNF 10 mg/kg treated group; each data is expressed in theaverage ±S.D. (n=5); *:P<0.05, **:P<0.01 vs. the vehicle-treated group(Dunnett's test).

As is shown in FIG. 3 and FIG. 4, like the effects on the blood glucoselevel, the effects of lecithinized BDNF on the amount of foodconsumption and the body weight were dose-dependent and more remarkablethan those of BDNF.

INDUSTRIAL APPLICABILITY

By modifying BDNF with a 1-acyl-glycerol derivative, there is obtained amodified BDNF having improved pharmacological activities,pharmacokinetics and the physical properties than BNDF. The BDNF beingmodified with a 1-acyl-glycerol derivative of the present invention isuseful as a therapeutic agent for treatment of neurodegenerativediseases. In addition, the present modified BDNF exhibits dose-dependenthypoglycemic activity as well as an activity of promoting reduction ofthe amount of food consumption and the body weight, and hence, it isuseful as a therapeutic agent for treatment of diabetes mellitus,especially type 2 diabetes mellitus.

What is claimed is:
 1. A modified brain-derived neurotrophic factorcomprising: a first moiety of a brain-derived neurotrophic factor, fromwhich a number average of 1 to 10 hydroxyl group(s) and/or hydrogenatom(s) have been removed from the carboxyl groups and/or amino groupsof amino acids of the brain-derived neurotrophic factor, and a secondmoiety that is a 1-acyl-glycerol derivative selected from the groupconsisting of 1-acyl-glycero-3-phosphoryl choline,1-acyl-glycero-3-phosphoryl serine and 1-acyl-glycero-3-phosphorylethylamine; wherein said first and second moieties are chemicallycross-linked.
 2. A modified brain-derived neurotrophic factor, whereinsaid modified brain-derived neurotrophic factor is a compound of formula(1): A(X—B)_(m)  (1) wherein A is a residue of a brain-derivedneurotrophic factor, B is a residue of a 1-acyl-glycerol derivativehaving an oxygen atom at the 2-position of the glycerol moiety, which isprepared by removing a hydrogen atom from the hydroxyl group at the2-position of the glycerol moiety, X is a chemical cross-linker, and mis number average of groups modifying the brain-derived neurotrophicfactor and ranges from 1 to about
 10. 3. The modified brain-derivedneurotrophic factor according to claim 2, wherein X of the modifiedbrain-derived neurotrophic factor is a group of the formula (2):

wherein R¹ is an alkylene group, or a group of the formula (3):

wherein R² and R³ are independently an alkylene group.
 4. The modifiedbrain-derived neurotrophic factor according to claim 2, wherein the1-acyl-glycerol derivative is 1-acyl-glycero-3-phosphoryl choline,1-acyl-glycero-3-phosphoryl serine, or 1-acyl-glycero-3-phosphorylethylamine.
 5. The modified brain-derived neurotrophic factor accordingto claim 2 or claim 3, wherein B of the modified brain-derivedneurotrophic factor is a group of the formula (4):

wherein R⁴ is an acyl group.
 6. The modified brain-derived neurotrophicfactor according to claim 4, wherein the acyl group of formula (4) is analkanoyl group having 8 to 30 carbon atoms.
 7. A pharmaceuticalcomposition comprising: a) a modified brain-derived neurotrophic factorof the formula (1): A(X—B)_(m)  (1) wherein A is a residue of abrain-derived neurotrophic factor, B is a residue of a 1-acyl-glycerolderivative having an oxygen atom at the 2-position of the glycerolmoiety, which is prepared by removing a hydrogen atom from the hydroxylgroup at the 2-position of the glycerol moiety, X is a chemicalcross-linker, and m is number average of groups modifying thebrain-derived neurotrophic factor and ranges from 1 to about 10; and b)a pharmaceutically acceptable carrier.
 8. The pharmaceutical compositionaccording to claim 7, wherein X of the modified brain-derivedneurotrophic factor is a group of the formula (2):

wherein R¹ is an alkylene group, or a group of the formula (3):

wherein R² and R³ are independently an alkylene group.
 9. Thepharmaceutical composition according to claim 7, wherein the1-acyl-glycerol derivative is 1-acyl-glycero-3-phosphoryl choline,1-acyl-glycero-3-phosphoryl serine, or 1-acyl glycero-3-phosphorylethylamine.
 10. The pharmaceutical composition according to claim 7 orclaim 8, wherein B of the modified brain-derived neurotrophic factor isa group of the formula (4):

wherein R⁴ is an acyl group.
 11. The pharmaceutical compositionaccording to claim 10, wherein the acyl group of formula (4) is analkanoyl group having 8 to 30 carbon atoms.
 12. A pharmaceuticallyacceptable salt of the modified brain-derived neurotrophic factor ofclaim
 2. 13. The modified brain-derived neurotrophic factor according toclaim 2, wherein the 1-acyl-glycerol derivative is lecithin.